In recent years, a method for evaluating the state of cells by conducting analysis on a per-cell basis has been drawing attention. Such a method is called single cell analysis, which is one of the important analytical methods conducted in sterile tests during manufacture of pharmaceuticals and in regenerative medicine typified by iPS (induced pluripotent stem) cells.
A measurement method that physically, chemically, or biologically analyzes an extremely small number of cells in a sample typically requires high sensitivity and precision. Therefore, for such type of measurement, a method for condensing cells, which are the measurement targets, using a filter by filtering a sample under a sterile environment and thus removing unnecessary mediums is used. There are also cases in which a filter with an analytical reagent added thereto is heated or cooled as needed in order to activate or inactivate reactions of the analytical reagent, or a filter is moved to an analyzer or the like.
In sterile tests, members that are used for filtering a sample, causing a reaction of a reagent, and moving an analyte should be in sterile conditions. Further, in sterile tests, it is required that not only a sample be easy to handle, but also contamination from a variety of contamination sources (e.g., an operator, measurement environment, or unnecessary samples remaining after filtration) be prevented. Furthermore, it is extremely important to prevent mutual contamination between samples, that is, cross-contamination in order to analyze a small number of valuable cells with high sensitivity and high-precision.
Hereinafter, the background art of a method for measuring bacterial cells using a bioluminescence method will be described. In the pharmaceutical manufacturing field, the cosmetic manufacturing field, the clinical medicine field, the basic biochemical field, and the like, determination of whether or not a sample contains bacterial cells and measurement of the number of bacterial cells are widely conducted in order to control quality of sample. For example, in the pharmaceutical manufacturing field, it is essential to manage bacterial cells (microbes or funguses) contained in the raw materials, intermediates, and end products of pharmaceuticals, or in pharmaceutical manufacturing water based on the Japanese pharmacopoeia standardized by the Japanese Ministry of Health, Labour and Welfare. Thus, the number of bacterial cells is measured each day.
The main method for measuring the number of microbes or funguses, which is standardized by the Japanese pharmacopoeia, is the culture method.
TABLE 1BioluminescenceFluorescentMethodStaining MethodCulture MethodDetection TargetsATP MoleculesCells containingCellsExtracted fromDNACellsDetection MethodLuminescentFluorescenceVisualReaction of ATP-Reaction betweenObservation ofLuciferaseDNA andColonyFluorescent DyeDo DetectionPassNot PassNot PassTargets Passthrough Filter?
In the culture method, a sample is first filtered through a filter to trap bacterial cells, and then, the filter that has trapped the bacterial cells is put on an agar plate for cultivation. At this time, a single bacterial cell forms a single colony. As the culture method, the number of colonies (CFU: colony forming unit) that can be measured is counted through visual observation using such characteristics, thereby quantitatively determining the count of viable bacteria in the sample. It should be noted that dead bacteria will not grow even when they are cultured, and will not form colonies. Thus, such bacteria are not visually observed.
By the way, in the pharmaceutical industry, bacterial cells under an oligotrophic environment, such as in pharmaceutical manufacturing water, are the detection targets. Therefore, a time as long as about one week is needed to culture such bacterial cells, which is problematic in detection. This, in turn, can take a long time to prepare test results in the stage of manufacturing intermediates or shipping final products. Thus, the culture method burdens the business operators both temporally and economically.
Meanwhile, the fluorescent staining method is known as a method that can rapidly measure bacterial cells. Patent Literature 1 describes an example of a microorganism collecting kit used in the fluorescent staining method. The microorganism collecting kit described in Patent Literature 1 is used by combining a filter for removing foreign matter as a pre-filter with a filter for collecting microorganisms, and is characterized in that the base main body of the filter is reusable.
In the latest fluorescent staining method, a sample containing bacterial cells is filtered first, and then, DNA (deoxyribonucleic acid) in the bacterial cells is stained using a fluorescent dye. There is known a method that uses two types of dyes: a fluorescent dye for concurrently staining DNA in viable bacteria and DNA in dead bacteria, and a fluorescent dye for staining only DNA in dead bacteria. Such a method can separately measure viable bacteria and dead bacteria.
However, in the fluorescent staining method, fine particles, dust, and the like other than bacterial cells would be concurrently detected if they emit fluorescence with the same wavelength as the fluorescent dye. Thus, the fluorescent staining method has a problem in that the reliability of the detection results of bacterial cells is unstable.
Besides, the ATP (adenosine triphosphate) bioluminescence method is known as a method for rapidly measuring bacterial cells using a principle other than fluorescence measurement. ATP molecules, which are the detection targets of this method, are organic compounds that exist in cells of all living organisms, and are the sources of energy that is necessary for vital activities of the cells. In the bioluminescence method, luciferase and luciferin that emit light upon chemically reacting with ATP are used, so that luminescence generated by a luminescent reaction between ATP extracted from cells and luciferase or luciferin is measured to estimate the number of cells from the amount of luminescence.
In the conventional method, a sample containing ATP derived from viable bacteria and dead bacteria as well as ATP in the free state is subjected to the following three stages: (1) removal of ATP other than ATP derived from viable bacteria, (2) extraction of ATP in the viable bacteria, and (3) a luminescent reaction between ATP derived from the viable bacteria and a luminous reagent (e.g., luciferase and luciferin) and measurement of the luminescence.
The amount of ATP contained in each viable bacterium is as small as about 1.5×10−18 mol/CFU (0.001 fmol/CFU=1 amol/CFU) when calculated in terms of 1 CFU of bacterium (Non Patent Literature 1). The ATP detection sensitivity of the currently available common bioluminescence method is 1×10−15 to 1×10−16 mol (1 to 0.1 fmol).